dNTP Imbalance and DNA Double Strand Breaks in Mouse FM3A Cells and the Mechanism of Cell Death

小鼠 FM3A 细胞中 dNTP 失衡和 DNA 双链断裂及细胞死亡机制

• 批准号: 05807206
• 负责人: WATAYA Yusuke
• 金额: $ 1.22万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05807206/
• 关键词: Cell Death; Endonuclease; DNA Fragmention; Apoptosis; Imbalance; DNA Double Strand Breaks; dNTP Pool; 5-Fluorodeoxyuridine; DNA二本切断; 分子機構; 制がん剤

The mechanism of deoxyribonucleoside triphosphate (dNTP) pool imbalanceinduced cell death in cultured mouse FM3A cells was studied. We reported previously that 5-fluorodeoxyuridine (FdUrd) can induce intracellular dNTP pool imbalance followed by DNA double-strand breaks and subsequent cell death. We have now found that the FdUrd-induced DNA fragments can be separated into two classes. One was large DNA fragments with sizes of 100-200 kbp, which corresponded to the replication units in mammalian genomes, and the other was shorter DNA fragments with a pitch of nucleosome length, characteristic for apoptosis. In addition, we have purified the double-strand break causing endonuclease from the lysate of FdUrd-treated FM3A cells. This endonuclease activity was detectable in the lysate of FdUrd-treated FM3A cells but not in untreated cells. The endonuclease was purified to near homogeneity and its molecular mass was estimated to be approximately 40 kDa by use of a DNA-containing SDS-PAGE.The endonuclease exhibited an optimal pH of 6.0-6.5 and did not require divalent metal cations for its activity. The cleavage of DNA by the endonuclease produced 5'-phosphoryl termini. Endonuclease of mammalian cells having these properties have not been described in the literature. We suspect that this endonuclease (which we termed Endonuclease S) plays an important role in the dNTP imbalance death, a process in many ways similar to 'apoptosis', the cellular suicide response.

本文研究了脱氧核苷三磷酸（dNTP）库失衡诱导小鼠FM 3A细胞死亡的机制。我们以前报道，5-氟脱氧尿苷（FdUrd）可以诱导细胞内dNTP池的失衡，随后的DNA双链断裂和随后的细胞死亡。我们现在已经发现，FdUrd诱导的DNA片段可以分为两类。一种是大小为100-200 kbp的大DNA片段，其对应于哺乳动物基因组中的复制单位，另一种是具有核小体长度的节距的较短DNA片段，其特征在于凋亡。此外，我们已经从FdUrd处理的FM 3A细胞的裂解物中纯化了引起双链断裂的核酸内切酶。这种核酸内切酶活性在FdUrd处理的FM 3A细胞的裂解物中可检测到，但在未处理的细胞中不可检测到。经纯化后的核酸内切酶分子量约为40 kDa，最适pH为6.0-6.5，不需要二价金属离子。核酸内切酶切割DNA产生5 ′-磷酰基末端。具有这些性质的哺乳动物细胞的核酸内切酶尚未在文献中描述。我们怀疑这种核酸内切酶（我们称之为核酸内切酶S）在dNTP失衡死亡中起着重要作用，这一过程在许多方面类似于“细胞自杀”，即细胞自杀反应。

项目成果

綿矢有佑: "制がん性ヌクレオシドによる細胞内DNA損傷の解析" 薬学研究奨励財団-研究成果報告. 10. 197-207 (1994)

Yusuke Wataya：“抗癌核苷引起的细胞内 DNA 损伤的分析”药物研究基金会 - 研究结果报告。 10. 197-207 (1994)。

J.W.Eckstein: "Mechanism-Based Inhibition of Thymidylatr Synthase by 5-Trifluoromethy1-2′-deoxyuridinr-5′-monophosphate" Biochemistry. 33. 15086-15094 (1994)

J.W.Eckstein：“5-三氟甲基1-2′-脱氧尿苷-5′-单磷酸对胸苷酸合酶的抑制机制”，《生物化学》33。15086-15094 (1994)

Y.Wataya, K.Takahashi1, T.Kakutani, K.Tanaka, and M.Nakamura: ""Induction of c-fos, c-jun and c-mic protooncogenes by treatment with Trp-P-2"." Mammalian Mutagenicity Study Group Communications. 7. 1-2 (1993)

Y.Wataya、K.Takahashi1、T.Kakutani、K.Tanaka 和 M.Nakamura：“用 Trp-P-2 处理诱导 c-fos、c-jun 和 c-mic 原癌基因”。

J.W.Eckstein, P.G.Foster, J.Fine-Moore, Y.Wataya, and D.V.Santi: ""Mechanism-Based Inhibition of Thymidylate Synthase by 5- Trifluoromethyl-2'-deoxyruidine-5'-monophosphate"." Biochemistry. 33. 15086-15094 (1994)

J.W.Eckstein、P.G.Foster、J.Fine-Moore、Y.Wataya 和 D.V.Santi：“5-三氟甲基-2-脱氧核苷-5-单磷酸对胸苷酸合成酶的基于机制的抑制”。

S.Takatori, A.Matsuda, J.Yamashita, H.Hayatsu, and Y.Wataya: ""Reacrion of 5-Trifluoromethy1-2'-deoxyuridine with Biswfite"." Nucleic Acids symposium Series. 31. 37-38 (1994)

S.Takatori、A.Matsuda、J.Yamashita、H.Hayatsu 和 Y.Wataya：“5-三氟甲基 1-2-脱氧尿苷与 Biswfite 的反应”。